Passing Cells and Counting and Plating S2 Cells for Plate 9 Transfection

Passing Dv-1 Cells

  • First I tried passing these cells with trypsin, but the cells didn’t release from the flask even after close to 10 minutes in trypsin
  • At that point I used a cell scraper to get the cells off the flask, and this worked well
  • The fluid was put in a 15mL tube
  • The tube was spun for 4 min at 800rpm
  • Removed the supernatant
  • Resuspended the cell pellet in 1mL 10% FBS Schneider’s medium
  • Used 100ul of that fluid to seed 2 new flasks, with 5mL of 10% FBS Schneider’s medium in each

Counting S2 Cells and Plating Them

  • I had a similar issue with using trypsin in these cells, the cells didn’t remove from the flask even in trypsin after a while, I again used a cell scraper on these cells, which worked fine
  • The fluid was put in a 15mL tube
  • The tube was spun for 4 min at 800rpm
  • Removed the supernatant
  • Resuspended the cell pellet in 1mL 10% FBS Schneider’s medium
  • 20ul of this cell suspention was put in a hemocyometer for counting:
section quadrant 1 quadrant 2 quadrant 3 quadrant 4 section average
1 183 234 253 197 217
2 229 194 167 178 192
  • Each section was averaged, and the average of the two sections was taken
    • Total S2 average: 205
  • The cells per mL is the average * 10^4
    • 205 * 10^4 = 2,050,000 cells per mL
  • (2.05*10^6 cells/mL)(1mL) = (150,000 cell/mL)(xmL)
    • x = 13mL medium
  • Because I only want ~5mL, I divided the xmL and the cell volume 1mL by 2
    • 6.5mL 10% FBS medium
    • 0.5mL cell resuspention
  • This was made, and 1.5mL S2 cell suspension was put in wells A1, B1, and C1

The plate was put in the incubator to grow overnight

Before adding the S2 cells, I imaged the 3.1 cells to see how they looked:

A2:

B2:

C2:

A3:

B3:

C3:

All of these look really good, maybe even too many cells! We will see how it goes for the transfection.